Our long term goal is to gain as complete an understanding as possible of the extracellular requirements for growth and differentiation of human mammary epithelial cells (HMEC) and also of the intracellular regulatory mechanisms that control those processes. The immediate goals of current phases of this program are to identify the extracellular signals that are needed for a high level of differentiation of HMEC in a defined medium, and to begin analyzing the control mechanisms that link these signals to selective expression of genes coding for milk proteins. During the preceding grant period, we focused on the development of research tools and needed for this research including the use of cloned cDNA and antisense RNA probes to analyze for human milk protein gene expression at the mRNA level, and antibodies to human milk proteins. During the first part of the proposed grant period, we will concentrate on obtaining high levels of expression of milk- related genes, and on precise identification of the control signals needed to achieve such expression under defined culture conditions. Special attention will be given to conditions that promote a polarized secretory cell morphology effects of hormones and matrix materials, effects of cellular interactions, and effects of the composition of the culture medium. Later, as specific regulatory signals are identified, we will determine the levels in the flow of information from DNA to final protein product at which they exert their effects. This will place us in a strong position to begin detailed studies of the intracellular regulatory mechanisms that link the extracellular signals to specific changes in gene expression. At the basic science level, these studies will allow advanced research that is now only possible with animal cells to be done with human material. In addition, this research will provide improved defined medium model systems for research on diverse aspects of human mammary cell biology and carcinogenesis. In particular, control over the differentiated state of the cells will allow studies on the protective effects of pregnancy (and perhaps also lactation) against human breast cancer to be undertaken using in vitro model systems.